Bromo-phenyl substituted thiazolyl dihydropyrimidines

ABSTRACT

This invention relates to a bromo-phenyl substituted thiazolyl dihydropyrimidine, its preparation method and use as a medicament for treating and preventing hepatitis B infections. The invention also relates to a composition comprising the dihydropyrimidine, one or more antiviral agents and, optionally, an immunomodulator for treating and preventing HBV infections.

This is a U.S. national stage application of the International PatentApplication No. PCT/CN2008/001187, filed Jun. 18, 2008, which claimspriority to Chinese Patent Application No. 200710119019.8, filed Jun.18, 2007, both of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to a new bromo-phenyl substituted thiazolyldihydropyrimidine, its preparation method and use as a medicamentespecially for treating and preventing hepatitis B infections. Theinvention also relates to a composition comprising thedihydropyrimidine, other antiviral agent and, when appropriate, animmunomodulator and a medicament comprising the composition especiallyfor treating and preventing HBV infections such as hepatitis Binfections.

BACKGROUND OF THE INVENTION

The hepatitis B virus belongs to the family of hepadna viruses. It cancause acute and/or persistent or progressive chronic diseases. Manyother clinical manifestations in the pathological state are also causedby the hepatitis B virus—in particular chronic inflammation of theliver, cirrhosis of the liver and hepatocellular carcinoma. In addition,coinfection with the hepatitis delta virus may have adverse effects onthe progress of the disease.

The interferon and lamivudine are conventional medicaments approved tobe used for treating chronic hepatitis. However, the interferon has justmoderate activity but has an adverse side reaction. Although lamivudinehas good activity, its resistance develops rapidly during the treatmentand relapse effects often appear after the treatment is stopped. TheIC₅₀ value of lamivudine (3-TC) is 300 nM (Science, 299 (2003),893-896).

U.S. Pat. No. 7,074,784 discloses 6-amidoalkyldihydropyrimidine and itsuse as a medicament especially for treating and preventing hepatitis Binfection.

It is described in Example 12 of U.S. Pat. No. 7,074,784 that R¹ iso-chlorine, R² is p-chlorine, R⁶ is 3,5-difluoro-pyridin-2-yl, X is—CH₂— and Z is morpholinyl. The compound can inhibit the growth ofhepatitis B virus during cell culturing. The IC₅₀ value is 2 nM (testedby themselves).

The main substitution in Example 12 is replacing bis-chlorine with R¹(o-bromine) and R² (p-fluorine), which results in the IC₅₀ of Compound 9being 7 nM (described in Example 9 of the patent). And when the mainsubstituents are changed into R¹ (o-chlorine) and R² (p-fluorine), anapproximate IC₅₀ value is also obtained (IC₅₀=2-4 nM in Example 5).

It is indicated that the IC₅₀ value cannot increase with the variationof the main substituents R¹ and R² (see Table 1).

U.S. Pat. No. 7,074,784 B2 also discloses an example, wherein a difluororesidue is substituted for thiazol-2-yl (described in Example 45 of thepatent). The derivative has a similar IC₅₀ value (2 nM) (see Table 1).

TABLE 1 Example 2 of U.S. Pat. No. 7,074,784 B2 Example R¹ R² R³ R⁶ IC₅₀(nM) 12 Cl Cl CH₃

2 (self-tested) 9 Br F CH₃

7 5 Cl F CH₃

2-4 45 Cl Cl CH₃

2

DETAILED DESCRIPTION OF THE INVENTION

We have surprisingly discovered that a derivative with an activity of 10times higher and the IC₅₀ value of less than 1 nM can be obtained bysubstituting with thiazol-2-yl and changing the main substituents intoR¹=o-bromine and R²=p-fluorine. This is unexpected when reading U.S.Pat. No. 7,074,784 (see Table 2).

TABLE 2 Some Examples of this Invention

Example R¹ R² R³ R⁶ IC₅₀ (nM) 1 Br F CH₃

0.3 2 Br Cl CH₂CH₃

0.2

This invention relates to a compound having formula (I) and its isomer(Ia),

wherein R¹ is o-bromine, R² is p-fluorine, R³ is C₁-C₄ alkyl, R⁶ isthiazol-2-yl, X is methylene and Z is morpholinyl.

Preferably, R¹ of the compound of the invention having formula (I) and(Ia) is o-bromine, R² is p-fluorine, R³ is methyl or ethyl, R⁶ isthiazol-2-yl, X is methylene and Z is morpholinyl.

This invention also relates to an enantiomer of the compound disclosedherein and a mixture thereof. The racemate can be separated by a knownmethod, and fundamentally it is a homogeneous component in astereoisomer mixture.

The compounds of the invention comprise an isomer having formula (I) and(Ia) and a mixture thereof.

The compound of the invention can also be in a form of a salt,preferably a physiologically acceptable salt.

The physiologically acceptable salt can be an inorganic acid salt ororganic acid salt. Preferably it is an inorganic acid salt such aschloride, bromide, phosphate or sulfate, etc., or a carboxylate or asulfonate, such as acetate, maleate, fumarate, malate, citrate,tartarate, lactate, benzoate or methanesulfonate, ethanesulfonate,benzenesulfonate, toluenesulfonate or naphthalenedisulfonate, etc.

The physiologically acceptable salt can also be a metal salt or anammonium salt of the compound of the invention. In a preferred example,it is a sodium salt, potassium salt, magnesium salt or calcium salt, andan ammonium salt produced by ammonia or organic amine such asethylamine, diethylamine or triethylamine, diethanolamine ortriethanolamine, dicyclohexylamine, dimethylaminoethyl alcohol,arginine, lysine, ethylenediamine or 2-phenylethylamine, etc.

The compound (I) of the invention can be prepared by the followingmethods:

[A] firstly a benzaldehyde having formula (II) reacts with a β-ketoesterhaving formula (III) with or without the addition of an alkali or anacid, and, when appropriate, in the presence of an inert organic solventto produce a benzylidene compound having formula (IV):

wherein R¹, R², R³, X and Z are as defined herein, and then thebenzylidene compound reacts with an amidine having formula (V) or a saltthereof (such as hydrochloride or acetate) with or without the additionof an alkali or an acid, and, when appropriate, in the presence of aninert organic solvent:

wherein R⁶ is as defined herein; or[B] the β-ketoester having formula (III) reacts with the benzaldehydehaving formula (II) and the amidine having formula (V) or a salt thereof(such as hydrochloride or acetate) with or without the addition of analkali or an acid, and, when appropriate, in the presence of an inertorganic solvent in one step; or[C] if X in formula (I) is methylene, a compound having formula (VI)reacts with morpholine having formula (VII) with or without the additionof an alkali, and, when appropriate, in the presence of an inert organicsolvent,

wherein R¹, R², R³ and R⁶ are as defined herein and Y is a nucleophilicsubstituent, such as chloro, bromo, iodo, methylsulfonyl ortoluenesulfonyl; or[D] the benzaldehyde having formula (II) reacts with a compound havingformula (X) and the amidine having formula (V) with or without theaddition of an alkali and, when appropriate, in an inert organicsolvent,

wherein R³, X and Z are as defined herein.

Compound of formula (VI) can be prepared by, for example, reacting acompound having formula (VIII)

wherein R¹, R², R³ and R⁶ are as defined herein, with a brominatingagent such as N-bromosuccinimide, preferably in an inert organicsolution, to produce a compound having formula (IX):

and reacting the compound having a nucleophilic substituent, directly orafter the compound being further converted according to a conventionalmethod as described in a literature, with the morpholine having formula(VII).

In order to prepare the compound of the invention having formula (I),wherein X is methylene and Z is morpholinyl, a chloroacetate havingformula (XI) reacts with morpholine (VII) to produce the β-ketocarboxylate of formula (III),

wherein R³ is as defined herein.

As a starting material, 2-bromo-4-fluoro-benzaldehyde (II) iscommercially available.

As a starting material, β-keto carboxylate (III) is well-known, or canbe prepared by known methods published in the literature [for example,D. Baumann, “Umsetzung von Diketen mit Alkoholen, Phenolen undMercaptanen”, in “Methoden der organischen Chemie” (Houben-Weyl), vol.VII/4, 230 ff (1968); Y. Oikawa, K. Sugano und O. Yonemitsu, J. Org.Chem. 43, 2087 (1978)].

The compound (V) is known and can be prepared according to thedescriptions of WO-A-99/54326 and WO-A-99/54329.

Morpholine (VII) is commercially available.

Compounds (VIII) and (X) can be prepared according to step [A] or [B]described in WO-A-99/54326.

All inert organic solvents are suitable for use in steps A, B, C and D.The inert organic solvent is preferably an alcohol such as methanol,ethanol and isopropyl alcohol, an ether such as dioxane, diethyl ether,tetrahydrofuran, ethylene glycol monomethyl ether, ethylene glycoldimethyl ether, a carboxylic acid such as acetic acid,dimethylformamide, dimethyl sulfoxide, acetonitrile, pyridine orhexamethyl phosphoric triamide.

The reaction temperature can be varied within quite a wide range.Usually the temperature is between 20° C. and 150° C. Preferably, thetemperature is the boiling temperature of the selected solvent.

The reaction can be carried out under the atmospheric pressure or undera high pressure. It is usually carried out under the atmosphericpressure.

The reaction can be carried out with or without an acid or alkali. It ispreferable to carry out the reaction in the presence of a weak acid suchas acetic acid, formic acid or the like.

An embodiment of the invention relates to a composition comprising A) atleast one of the above dihydropyrimidines and B) at least one of otherantiviral agents different from A).

A certain embodiment of the invention relates to a compositioncomprising A) the above dihydropyrimidine, B) an HBV polymeraseinhibitor and, when appropriate, C) an immunomodulator.

Preferably the immunomodulator C) is selected from, for example, all theinterferons such as α-interferon, β-interferon and γ-interferon,especially α-2a-interferon and α-2b-interferon, an interleukin such asinterleukin-2, a polypeptide such as thymosin-α-1 and a thymoctonan, animidazoquinoline derivative such as levamisole, an immunoglobulin and atherapeutic vaccine.

Thereby, this invention also relates to a composition for treating andpreventing HBV infections and its use for treating diseases induced byHBV.

The use of the combinations of the invention provides valuableadvantages for the treatment of HBV-induced diseases compared withmonotherapy with the individual compounds, namely principally asynergistic antiviral activity, but also good tolerability of thecombinations of the invention in Tox-50 (the range of toxicity at which50% of the cells survive).

The substances referred to as HBV polymerase inhibitors B for thepurposes of the invention are those which, in the endogenous polymeraseassay which was published by Ph. A. Furman et al. in AntimicrobialAgents and Chemotherapy, Vol. 36 (No. 12), 2688 (1992) and which isdescribed hereinafter, lead to an inhibition of the formation of an HBVDNA double strand, so as to result in a maximum of 50% of the activityof the zero value.

HBV polymerase inhibitors B for use in the invention are the substancesdisclosed in the endogenous polymerase experiment published in“Antimicrobial Agents and Chemotherapy” Vol. 36 (No. 12), 2688 (1992) byPh. A. Furman, and the substances described below for inhibiting theformation of double-stranded HBV DNA thereby resulting in the maximum50% activity value to be zero.

HBV virions from culture supernatants incorporate nucleoside5′-triphosphates into the plus strand of the HBV DNA in vitro. By usingagarose gel electrophoresis, the incorporation of[α-³²P]-deoxynucleoside 5′-triphosphate into the viral 3.2 kb DNAproduct is observed in the presence and absence of a substancepotentially having HBV polymerase-inhibiting properties. HBV virions areobtained from the cell culture supernatant of HepG2.2.15 cells byprecipitation with polyethyleneglycol and are concentrated. One part byvolume of clarified cell culture supernatant is mixed with ¼ by volumeof an aqueous solution containing 50% by weight polyethylene glycol 8000and 0.6 M sodium chloride. The virions are sedimented by centrifugationat 2500×g/15 minutes. The sediments are resuspended in 2 ml of buffercontaining 0.05 M tris-HCl (pH 7.5) and dialyzed against the same buffercontaining 100 mM potassium chloride. The samples can be frozen at −80°C. Each reaction mixture (100 μl) contains at least 105 HBV virions; 50mM tris-HCl (pH 7.5); 300 mM potassium chloride; 50 mM magnesiumchloride; 0.1% Nonident® P-40 (nonionic detergent from BoehringerMannheim); 10 μM dATP, 10 μM dGTP, 10 μM dTTP; 10 μCi [³²P]dCTP (3000Ci/mmol; final concentration 33 nM) and 1 μM of the potential polymeraseinhibitor in its triphosphorylated form. The samples are incubated at37° C. for one hour and then the reaction is stopped by adding 50 mMEDTA. A 10% weight/volume SDS solution (containing 10 g of SDS per 90 mlof water) is added to a final concentration of 1% by volume (based onthe total volume), and proteinase K is added to a final concentration of1 mg/ml. After incubation at 37° C. for one hour, samples are extractedwith the same volume of phenol/chloroform/isoamyl alcohol (ratio 25:24:1by volume), and the DNA is precipitated from the aqueous phase withethanol. The DNA pellet is resuspended in 10 μl of gel buffer (solutionof 10.8 g of tris, 5.5 g of boric acid and 0.75 g of EDTA in 1 liter ofwater (=TBE buffer)) and separated by electrophoresis in an agarose gel.Either the gel is dried or the nucleic acids present therein transferredby the Southern transfer technique to a membrane. The amount of labeledDNA double strand formed is then determined in relation to the negativecontrol (=endo-pol reaction without substance or with inactive controlsubstance). An HBV polymerase inhibitor is present if a maximum of 50%of the activity of the negative control is present.

Preferred HBV polymerase inhibitors B) comprise, for example,3TC=lamivudine=4-amino-1-[(2R-cis)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl-]-pyrimidin-2(1H)-one,cf. EP-B 382 526 (=U.S. Pat. No. 5,047,407) and WO 91/11186 (=U.S. Pat.No. 5,204,466); Adefovirdipivoxil=9-{2-[[bis[(pivaloyloxy)-methoxy]-phosphinyl]-methoxy]-ethyl}-a-denine,cf. EP-B 481 214 (=U.S. Pat. Nos. 5,663,159 and 5,792,756), U.S. Pat.Nos. 4,724,233 and 4,808,716; BMS200475=[1S-(1-α,3-α,4-β)]-2-amino-1,9-dihydro-9-[4-hydroxy-3-(hydroxymethyl)-2-methylene-cyclopentyl]-6H-purin-6-one,cf. EP-B 481 754 (=U.S. Pat. Nos. 5,206,244 and 5,340,816), WO 98/09964and 99/41275;Abacavir=(−)-(1S-cis)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol,cf. EP-B 349 242 (=U.S. Pat. No. 5,049,671) and EP-B 434 450 (=U.S. Pat.No. 5,034,394);FTC=(2R-cis)-4-amino-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-pyrimidin-2(1H)-one,cf. WO 92/14743 (=U.S. Pat. Nos. 5,204,466, 5,210,085, 5,539,116,5,700,937, 5,728,575, 5,814,639, 5,827,727, 5,852,027, 5,892,025,5,914,331, 5,914,400) and WO 92/18517;β-L-FDDC=5-(6-amino-2-fluoro-9H-purin-9-yl)-tetrahydro-2-furanmethanol,cf. WO 94/27616 (=U.S. Pat. Nos. 5,627,160, 5,561,120, 5,631,239 and5,830,881);L-FMAU=1-(2-deoxy-2-fluoro-β-L-arabinofuranosyl)-5-methyl-pyrimidine-2,4(1H,3H)-dione,cf. WO 99/05157, WO 99/05158 and U.S. Pat. No. 5,753,789.

A further preferred embodiment of the invention relates to a compositioncomprising A) the above dihydropyrimidines having formula (I) and (Ia);and B) lamivudine.

Other preferred HBV antiviral agents B comprise, for example,phenylpropenamides of the following formula:

wherein R¹ and R² are, each independently, C₁₋₄ alkyl or, together withthe nitrogen atom on which they are located, form a ring having 5 to 6ring atoms which comprise carbon and/or oxygen; R³ to R¹² are eachindependently hydrogen, halogen, C₁₋₄ alkyl, optionally substituted C₁₋₄alkoxy, nitro, cyano or trifluoromethyl; and R¹³ is hydrogen, C₁₋₄alkyl, C₁₋₇ acyl or aralkyl and X is halogen or optionally substitutedC₁₋₄ alkyl.

The phenylpropenamides and their preparation methods are disclosed in WO98/33501, and are mentioned here for publication. AT-61 is the compound

Preferred immunomodulators C) comprise, for example, all interferonssuch as α-, β- and γ-interferons, in particular also α-2a- andα-2b-interferons, interleukins such as interleukin-2, polypeptides suchasthymosin-α-1 and thymoctonan, imidazoquinoline derivatives such asLevamisole®, immunoglobulins and therapeutic vaccines.

A further preferred embodiment of the invention relates to combinationsof A) above dihydropyrimidines (I) and (Ia), B) lamivudine and, whereappropriate, C) an interferon.

Description of Tests

The antiviral action of the compounds of the invention on hepatitis Bvirus is investigated by methods based on those described by M. A. Sellset al., Proc. Natl. Acad. Sci., 84, 1005-1009 (1987) and B. E. Korba etal., Antiviral Research 19, 55-70 (1992).

The antiviral tests are carried out in 96-well microtiter plates. Thefirst vertical row of the plate receives only growth medium andHepG2.2.15 cells. It serves as virus control.

Stock solutions of the test compounds (50 mM) are initially dissolved inDMSO, and further dilutions are prepared in the HepG2.2.15 growthmedium. The compounds according to the invention are usually pipetted ina test concentration of 100 μM (1st test concentration) in each caseinto the second vertical test row of the microtiter plate andsubsequently diluted in twofold steps 210 times in growth medium plus 2%by weight of fetal calf serum (volume 25 μl)

Each well of the microtiter plate then contains 225 μl of HepG2.2.15cell suspension (5×104 cells/ml) in growth medium plus 2% by weight offetal calf serum. The test mixture is incubated at 37° C. and 5% CO2(v/v) for 4 days.

The supernatant is then aspirated off and discarded, and the wellsreceive 225 μl of freshly prepared growth medium. The compoundsaccording to the invention are each added anew as 10-fold concentratedsolution in a volume of 25 μl. The mixtures are incubated for a further4 days

Before harvesting the supernatants to determine the antiviral effect,the HepG2.2.15 cells are examined under the light microscope or by meansof biochemical detection methods (for example Alamar Blue stain orTrypan Blue stain) for cytotoxic changes

The supernatant and/or cells are then harvested and sucked by means of avacuum onto 96-well dot-blot chambers covered with a nylon membrane (inaccordance with the manufacturer's information).

Cytotoxicity Determination

Substance-induced cytotoxic or cytostatic changes in the HepG2.2.15cells are detected, for example, under the light microscope as changesin cell morphology. Such substance-induced changes in the HepG2.2.15cells compare with untreated cells are visible, for example, ascytolysis, vacuolation or altered cell morphology. A 50% cytotoxicity(Tox.-50) means that 50% of the cells show a morphology comparable tothe corresponding cell control.

The tolerability of some of the compounds according to the invention isadditionally tested on other host cells such as, for example, HeLacells, primary human peripheral blood cells or transformed cell linessuch as H-9 cells.

No cytotoxic changes are detectable at concentrations >10 μM of thecompounds of the invention.

Determination of the Antiviral Action

After the supernatants or lysed cells is transferred to the nylonmembrane of the blot apparatus (see above), the intra- or extracellularsupernatants of the HepG2.2.15 cells are denatured (1.5 M NaCl/0.5 NNaOH), neutralized (3 M NaCl/0.5M Tris HCl, pH 7.5) and washed (2×SSC).The DNA is then baked onto the membrane by incubating the filters at120° C. for 2-4 hours.

DNA Hybridization

Detection of the viral DNA from the treated HepG2.2.15 cells on thenylon filters is usually carried out with nonradioactive,digoxigenin-labeled hepatitis B-specific DNA probes, each of which islabeled with digoxigenin, purified and employed for the hybridization inaccordance with the manufacturer's information.

The prehybridization and hybridization take place in 5×SSC, 1× blockingreagent, 0.1% by weight N-lauroylsarcosine, 0.02% by weight SDS and 100μg of herring sperm DNA. The prehybridization takes place at 60° C. for30 minutes, and the specific hybridization takes place with 20 to 40ng/ml of the digoxigenized, denatured HBV-specific DNA (14 hours, 60°C.). The filters are then washed.

Detection of HBV-DNA by Digoxigenin Antibodies

The immunological detection of the digoxigenin-labeled DNA took place inaccordance with the manufacturer's information:

The filters were washed and prehybridized in a blocking reagent (inaccordance with the manufacturer's information). Hybridization was thencarried out with an anti-DIG antibody coupled to alkaline phosphatasefor 30 minutes. After a washing step, the substrate of alkalinephosphatase, CSPD, was added, incubated with the filters for 5 minutes,then packed in plastic film and incubated at 37° C. for a further 15minutes. The chemiluminescence of the hepatitis B-specific DNA signalswas visualized by exposing the filters to an X-ray film (incubationdepending on signal strength: 10 minutes to 2 hours).

The half-maximum inhibitory concentration (IC₅₀, 50% inhibitoryconcentration) was determined as the concentration at which the intra-or extracellular hepatitis B-specific band was reduced by the compoundaccording to the invention by 50% compared with an untreated sample.

It is unexpected that the compound of the invention exhibits aneffective antiviral effect with an IC₅₀ less than 1 nM. Therefore, thecompound of the invention is suitable for use in treating the diseasesinduced by viruses, especially acute and chronic persistent HBVinfections. Chronic viral diseases induced by HBV can worsen themorbidity and the chronic hepatitis B virus infection can cause livercirrhosis and/or hepatocellular carcinoma in many cases.

Areas of indication which may be mentioned for the compounds of theinvention are, for example: the treatment of acute and chronic viralinfections which may lead to infectious hepatitis, for exampleinfections with hepatitis B viruses. The compounds of the invention areparticularly suitable for the treatment of chronic hepatitis Binfections and the treatment of acute and chronic hepatitis B viralinfections.

The present invention includes pharmaceutical preparations which,besides nontoxic, inert pharmaceutically suitable carriers, comprise oneor more compounds (I) or (Ia) or a combination of the invention or whichconsist of one or more active ingredients (I) or (Ia) or of acombination of the invention.

The active ingredients (I) and (Ia) are intended to be present in thepharmaceutical preparations mentioned above in a concentration of about0.1 to 99.5% by weight, preferably of about 0.5 to 95% by weight, of thecomplete mixture.

The pharmaceutical preparations mentioned above may also comprise otheractive pharmaceutical ingredients apart from the compounds (I) and (Ia).

The ratio of the amounts of the components A, B and, where appropriate,C in the compositions of the invention may vary within wide limits; itis preferably 5 to 500 mg of A/10 to 1000 mg of B, in particular 10 to200 mg of A/20 to 400 mg of B.

Component C, which is also to be used where appropriate, may be used inamounts of, preferably, 1 to 10 million, in particular 2 to 7 million,I.U. (international units), about three times a week over a period of upto one year.

The compounds or compositions of the invention are intended to bepresent in the pharmaceutical preparations mentioned above in general ina concentration of about 0.1 to 99.5, preferably about 0.5 to 95, % byweight of the complete mixture.

The pharmaceutical preparations mentioned above can be produced in aconventional way by known methods, for example by mixing the activeingredient(s) with the carrier(s).

It has generally proved to be advantageous both in human and inveterinary medicine to administer the active ingredient(s) in totalamounts of about 0.5 to about 500, preferably of 1 to 100 mg/kg of bodyweight every 24 hours, where appropriate in the form of a plurality ofsingle doses, to achieve the desired results. A single dose contains theactive ingredient(s) preferably in amounts of about 1 to about 80, inparticular 1 to 30 mg/kg of body weight. However, it may be necessary todeviate from the dosages mentioned, in particular depending on thespecies and body weight of the subject to be treated, the nature andseverity of the disorder, the type of preparation and mode ofadministration of the medicament, and the time or interval within whichadministration takes place.

The invention therefore relates further to the compounds andcompositions defined above for controlling diseases.

The invention further relates to medicaments comprising at least one ofthe compounds or compositions defined above and, where appropriate, oneor more other active pharmaceutical ingredient(s).

The invention further relates to the use of the compounds andcompositions defined above for producing a medicament for the treatmentand prophylaxis of the diseases described above, preferably of viraldiseases, in particular of hepatitis B.

The percentage data in the following examples relate in each case toweight unless indicated otherwise. The ratios of solvents in solventmixtures are in each case based on volume.

EXAMPLES A. Preparation of Intermediates Intermediate 1 Ethyl4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-6-methyl-1,4-dihydropyrimidine-5-carboxylicester

A mixture of 10.0 g (49.3 mmol) of 2-bromo-4-fluorobenzaldehyde, 6.4 g(49.3 mmol) of ethyl acetoacetate, 8.1 g (49.3 mmol) of2-amidino-thiazole hydrochloride and 4.8 g (58.5 mmol) of sodium acetatewas dissolved or suspended in 400 ml of ethanol and then boiled andrefluxed for 16 hours. The solution obtained was cooled to roomtemperature and filtered. The residue was washed with water to removeinorganic salts. The product of 10.8 g (51.6%) was obtained. Meltingpoint: 163-165° C.

Intermediate 2 Methyl4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-6-methyl-1,4-dihydropyrimidine-5-carboxylicester

Intermediate 2 was synthesized from methyl acetoacetate by a methodsimilar to that for Intermediate 1. Yield: 53% (melting point: 155-157°C.).

Intermediate 3 Ethyl6-bromomethyl-4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylicester

5.0 g (11.8 mmol) of Intermediate 1 was added into 100 ml of carbontetrachloride and was heated to 50° C. in an atmosphere of the argon gasto obtain a clear solution. At this temperature, 2.33 g (13.0 mmol) ofN-bromosuccinimide was added into the solution and mixed at thetemperature for 10 minutes. The solution obtained was then cooledimmediately and filtered at room temperature, and decompressed forconcentration. The product obtained has a purity of higher than 90%according to the test result of HPLC, and was used as a raw material inthe next step. Rf=0.69 (the ratio of petroleum ether to ethyl acetate is8:2).

Intermediate 4 Methyl6-bromomethyl-4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-1,4-dihydropyrimidine-5-carboxylicester

Intermediate 4 was synthesized from Intermediate 2 by a method similarto that for the preparation of Intermediate 3. Rf=0.69 (the ratio ofpetroleum ether to ethyl acetate is 8:2).

B. Preparations of Examples Example 5 Ethyl4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-6-(4-morpholinylmethyl)-1,4-dihydropyrimidine-5-carboxylicester

2.0 g of Intermediate 2 was added into 15 ml of methanol to form asolution. The solution was mixed with 5 times of morpholine and stirredfor 30 minutes at room temperature. The solution obtained was thendiluted with water and extracted with ethyl acetate. Yield: 1.7 g.Melting point: 161-163° C. Rf=0.45 (the ratio of petroleum ether toethyl acetate is 8:2)

Example 6 Methyl4-(2-bromo-4-fluorophenyl)-2-(thiazol-2-yl)-6-(4-morpholinylmethyl)-1,4-dihydropyrimidine-5-carboxylicester

Example 6 was synthesized from Intermediate 4 by a method similar tothat for the preparation of Example 5. Melting point: 173-175° C.Rf=0.43 (the ratio of petroleum ether to ethyl acetate is 8:2).

The enantiomers prepared in Example 5 and Example 6 were separated on achiral column (Daicel Chiralpak AS-H, mobile phase:n-hexane/ethanol=99/1).

The anti-HBV active compounds in the two examples are enantiomers havinga relatively long retention time.

The activity data of the compounds of the invention are listed below:

Example No. IC₅₀ (nM) 5 0.2 (—)-5 0.1 6 0.3 (—)-6 0.2

The treatment of the hepatitis B virus-producing HepG2.2.15 cells withthe compounds of the invention can lead to a reduction in intra- and/orextracellular viral DNA.

INDUSTRIAL APPLICABILITY

The examples disclosed herein show that the compounds disclosed hereinexhibit an effective antiviral effect with the IC₅₀ less than 1 nM.Therefore, the compounds can be used for the treatment of a diseaseinduced by viruses, especially acute and chronic persistent HBVinfections according to the methods of the invention or any method knownto a person skilled in the art.

1. A compound having formula (I) or its isomer (Ia):

or an enantiomer or a salt thereof, wherein R¹ is o-bromine, R² isp-fluorine, R³ is a C₁-C₄ alkyl, R⁶ is thiazolyl-2-yl, X is methylene,and Z is morpholinyl.
 2. The compound of claim 1 or the enantiomer orthe salt thereof, wherein R¹ is o-bromine, R² is p-fluorine, R³ ismethyl or ethyl, R⁶ is thiazolyl-2-yl, X is methylene, and Z ismorpholinyl.
 3. A compound having one of the following structures or anenantiomer, tautomer or salt thereof:


4. A compound having one of the following structures or a levo isomer,tautomer or salt thereof:


5. The compound according to claim 1 or the enantiomer or the saltthereof, wherein the salt is an inorganic acid salt or an organic acidsalt.
 6. The compound according to claim 5 or the enantiomer or the saltthereof, wherein the inorganic acid salt is a hydrochloric acid salt, ahydrobromic acid salt, a phosphoric acid salt or a sulfuric acid salt.7. The compound according to claim 5 or the enantiomer or the saltthereof, wherein the organic acid salt is a carboxylate or a sulfonate.8. The compound according to claim 7 or the enantiomer or the saltthereof, wherein the carboxylate is acetate, maleate, fumarate, malate,citrate, tartarate, lactate or benzoate.
 9. The compound according toclaim 7 or the enantiomer or the salt thereof, wherein the sulfonate ismethanesulfonate, ethanesulfonate, benzenesulfonate, toluenesulfonate ornaphthalenedisulfonate.
 10. A method of preparing the compound of claim1, wherein the method is characterized by: (a) reacting a benzaldehydehaving formula (II) with a β-ketoester having formula (III) to produce abenzylidene compound having formula (IV):

and (b) reacting the benzylidene compound having formula (IV) with anamidine having formula (V):

or a salt thereof wherein R¹ is o-bromine, R² is p-fluorine, R³ is aC₁-C₄ alkyl, R⁶ is thiazolyl-2-yl, X is methylene, and Z is morpholinyl.11. A method of preparing the compound of claim 1, wherein the method ischaracterized by reacting a compound having formula (III) with analdehyde having formula (II) and an amidine having formula (V) or a saltthereof in one step,

wherein R¹ is o-bromine, R² is p-fluorine, R³ is a C₁-C₄ alkyl, R⁶ isthiazolyl-2-yl, X is methylene, and Z is morpholinyl.
 12. A method ofpreparing the compound of claim 1, wherein X of formula (I) is methyleneand the method is characterized by reacting the compound having formula(VI) with morpholine (VII) or a salt thereof:

wherein Y is a nucleophilic substituent, and R¹ is o-bromine, R² isp-fluorine, R³ is a C₁-C₄ alkyl, and R⁶ is thiazolyl-2-yl.
 13. A methodof preparing the compound of claim 1, which is characterized by the stepof reacting a compound having formula (II) with an aldehyde havingformula (X) and an amidine having formula (V) or a salt thereof:

wherein R¹ is o-bromine, R² is p-fluorine, R³ is a C₁-C₄ alkyl, R⁶ isthiazolyl-2-yl, X is methylene, and Z is morpholinyl.
 14. A compositioncomprising the following components: A) at least one compound accordingto claim 1; B) at least an HBV antiviral agent which is different fromcomponent A; and, when appropriate, C) at least an immunomodulator or aninterferon.
 15. The composition of claim 14, wherein the component B isan HBV polymerase inhibitor, lamivudine or a phenylpropenamide compoundhaving the following formula:

or a salt thereof, wherein each of R¹ and R² is independently C₁₋₄ alkylor, together with the nitrogen atom on which they are located, form aring having 5 to 6 ring atoms which comprise carbon and/or oxygen; andeach of R³ to R¹² is independently hydrogen, halogen, C₁-C₄-alkyl,optionally substituted C₁-C₄-alkoxy, nitro, cyano or trifluoromethyl.16. The composition of claim 15, wherein the component B is thephenylpropenamide compound having the following structure:


17. A medicament comprising at least one composition of claim 14, and,when appropriate, one or more active pharmaceutical agents.
 18. A methodfor treating hepatitis B infection or a disease caused by hepatitis Binfection, which comprises administering the composition of claim 14 toa patient having the disease.
 19. The method of claim 18, wherein themethod is for treating the disease caused by hepatitis B infectionselected from hepatitis, cirrhosis or hepatocellular carcinoma.
 20. Amethod for treating hepatitis B infection or a disease caused byhepatitis B infection, which comprises administering the compound ofclaim 1 to a patient having the disease.
 21. The method of claim 20,wherein the method is for treating the disease caused by hepatitis Binfection selected from hepatitis, cirrhosis or hepatocellularcarcinoma.
 22. A pharmaceutical composition comprising at least onecompound according to claim 1, and, when appropriate, a pharmaceuticallyacceptable carrier.